Human mass balance study of the novel anticancer agent ixabepilone using accelerator mass spectrometry.

Ixabepilone (BMS-247550) is a semi-synthetic, microtubule stabilizing epothilone B analogue which is more potent than taxanes and has displayed activity in taxane-resistant patients. The human plasma pharmacokinetics of ixabepilone have been described. However, the excretory pathways and contribution of metabolism to ixabepilone elimination have not been determined. To investigate the elimination pathways of ixabepilone we initiated a mass balance study in cancer patients. Due to autoradiolysis, ixabepilone proved to be very unstable when labeled with conventional [14C]-levels (100 microCi in a typical human radio-tracer study). This necessitated the use of much lower levels of [14C]-labeling and an ultra-sensitive detection method, Accelerator Mass Spectrometry (AMS). Eight patients with advanced cancer (3 males, 5 females; median age 54.5 y; performance status 0-2) received an intravenous dose of 70 mg, 80 nCi of [14C]ixabepilone over 3 h. Plasma, urine and faeces were collected up to 7 days after administration and total radioactivity (TRA) was determined using AMS. Ixabepilone in plasma and urine was quantitated using a validated LC-MS/MS method. Mean recovery of ixabepilone-derived radioactivity was 77.3% of dose. Fecal excretion was 52.2% and urinary excretion was 25.1%. Only a minor part of TRA is accounted for by unchanged ixabepilone in both plasma and urine, which indicates that metabolism is a major elimination mechanism for this drug. Future studies should focus on structural elucidation of ixabepilone metabolites and characterization of their activities.

Quantitative determination of butorphanol and its metabolites in human plasma by gas chromatography-electron capture negative-ion chemical ionization mass spectrometry.

Two separate analytical methods have been developed for the determination of butorphanol and its metabolites in human plasma. One method is specific for butorphanol (I) while the other determines the metabolites, hydroxybutorphanol (II) and norbutorphanol (III). Both procedures incorporate solid-phase extraction, chemical derivatization and separation, and detection using gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry (GC-ECNCI-MS). Both methods use the cyclopropyl analog of I (BC-2605, IV) as the internal standard and the procedures for extraction of the analytes from plasma are identical. However, following extraction, either the pentafluorobenzoyl ester of I or the tris and bis-trifluoroacetyl esters of II and III, respectively, were prepared. The derivatives were analyzed by GC-ECNCI-MS with selected-ion monitoring of the molecular ions. The standard curves were linear over the concentration ranges of 20-2000, 20-1000 and 50-1000 pg/ml for I, II and III, respectively. All standard curves from the assay validation had r2 values of > or = 0.994, 0.991 and 0.985 for I, II and III, respectively. For all three compounds, the intra- and inter-assay precisions (C.V.) and inter-assay accuracy (deviation from nominal) were within 12% for plasma quality control samples. All derivatives were stable in the reconstitution solvent for at least 24 h. The assays are being used for the determination of plasma concentrations of I, II and III in humans following repeated administration of nasal spray.

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection.

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH4)2HPO4, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH4)2HPO4, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5-500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at -20 degrees C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection.

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile-methanol-water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-microns column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1-100 ng/ml for butorphanol and hydroxybutorphanol, and 2-200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma.

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 microns column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10-1000 ng/ml (0.012-1.17 microM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.

High-performance liquid chromatographic method for the determination of nefazodone and its metabolites in human plasma using laboratory robotics.

A quantitative analytical method, using high-performance liquid chromatography and ultraviolet detection, has been established for the determination of nefazodone (NEF) and its metabolites, m-chlorophenylpiperazine (mCPP),p-hydroxynefazodone (PHN), and hydroxynefazodone (HO-NEF), in human plasma. The fully automated, robotic procedure consisted of addition of internal standard (aprindine), extraction with butyl chloride, followed by phase separation, organic phase evaporation, reconstitution of the residue, and injection onto the chromatographic system. The limits of detection for NEF, mCPP, PHN, and HO-NEF were 5, 1, 10, and 5 ng/ml, respectively, at a signal-to-noise ratio of 4. The method had a linear range of 10-1000 ng/ml for NEF and HO-NEF, 20-2000 ng/ml for PHN, and 2.5-250 ng/ml for mCPP. Correlation coefficients of 0.996 or greater were obtained during validation and study sample analysis.

Simultaneous quantitation of buspirone and 1-(2-pyrimidinyl)piperazine in human plasma and urine by capillary gas chromatography-mass spectrometry.

Buspirone and a buspirone metabolite, 1-(2-pyrimidinyl)piperazine (1-PP), are extracted from matrix using C18 extraction columns. The metabolite and its internal standard (d4-1-PP) are derivatized with pentafluorobenzoyl chloride to the corresponding amides. The 1-PP derivatives, buspirone and the buspirone internal standard (5-fluorobuspirone) are co-chromatographed. Chromatography and detection are performed using capillary gas chromatography with a fused-silica column and selected-ion monitoring-mass spectrometry. Linear range of the standard curves in plasma is 0.1-14 ng/ml for buspirone and 0.2-25 ng/ml for 1-PP with lower limits of quantitation of 0.1 and 0.2 ng/ml, respectively. In urine the linear range of the standard curves is 0.2-14 ng/ml for buspirone and 8-500 ng/ml for 1-PP with lower limits of quantitation of 0.2 and 8.0 ng/ml, respectively. Intra-assay accuracies were within 14% for buspirone and 1-PP in plasma and urine. Intra-assay precision was within 12% for both compounds in both matrices.

Liquid chromatographic procedure for the quantitative analysis of carboplatin in beagle dog plasma ultrafiltrate.

A specific and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of carboplatin (JM-8) in dog plasma ultrafiltrate. Plasma ultrafiltrate samples were generated using Amicon Centrifree micropartition systems or Amicon Centriflo cones, and injected onto a microBondapak NH2 column. The mobile phase consisted of acetonitrile:methanol:0.005 M sodium perchlorate, pH 2.4 (77-75:13-15:10, v/v/v); the flow rate was 1.5 mL/min. Detection was performed by monitoring UV absorbance of the column effluent at 229 nm. Carboplatin eluted between 9.5 and 11.0 min. The internal standard, JM-10, eluted between 11.0 and 13.0 min. The peak height ratio of carboplatin:internal standard versus carboplatin concentration was linear over a range of 0.2 to 20.0 micrograms/mL. The limit of quantitation was 0.2 microgram/mL. The intra-assay precision of this method, as measured by percent relative standard deviation (%RSD), was within 12% for the theoretical concentrations 0.5, 5.0, and 50.0 micrograms/mL. Accuracies were within 11%. The results of the validation procedures indicated that this procedure was accurate and specific.

The disposition of carboplatin in the beagle dog.

Carboplatin was administered i.v. to four groups of three male beagle dogs at doses of 3, 6, 12, and 24 mg/kg (60-580 mg/m2). Plasma samples were obtained at appropriate times and protein-free plasma ultrafiltrates (PU) were generated with Amicon Centrifree micropartition systems. Urine was collected at 24-h intervals for 96 h. PU and urine samples were analyzed for carboplatin by HPLC and for total platinum by atomic absorption spectrophotometry. Carboplatin accounted for about 90% of the free platinum in plasma. The Cmax and AUCinf values for carboplatin and for free platinum increased linearly with dose. The terminal elimination half-life and mean residence times for carboplatin and free platinum were each about 1 h. Total-body clearances for carboplatin (5.6 l/h per m2) and free platinum (5.1 l/h per m2) were constant over the dose range studied, as were the respective volumes of distribution (5.7 and 5.0 l/m2). A mean of 46% of the dose was excreted as carboplatin in 24-h urine; and by 72 h, 70% of the platinum administered was excreted in the urine. Free platinum was cleared by both renal and non-renal processes. These results show that a dose of carboplatin is rapidly excreted in the urine and that carboplatin and plasma-free platinum exhibit linear pharmacokinetics in the beagle dog.

High-performance liquid chromatographic method for the determination of etoposide in plasma using electrochemical detection.

A quantitative analytical method has been established for the determination of a semi-synthetic epipodophyllotoxin, etoposide, in plasma. The method employs reversed-phase high-performance liquid chromatography and electrochemical detection. Sample preparation consisted of extraction with 1,2-dichloroethane followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Observed recoveries were 76.8 and 87.5% for 50 and 500 ng/ml, respectively. The method had a linear range of 10-1000 ng/ml. Correlation coefficients of 0.997 or greater were obtained during validation experiments and study sample analysis.

Disposition of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-phosphate in mice and dogs.

The metabolic disposition of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-phosphate (2-F-araAMP) has been studied in mice and dogs after iv administration. Following injection of 40 mg/m2 into mice, serum levels of 2-F-araAMP fell, with apparent half-lives of 0.7 mins for the alpha phase and 21 mins for the beta phase. Rapid dephosphorylation of the compound resulted in high levels of the nucleoside, 9-beta-D-arabinofuranosyl-2-fluoroadenine (2-F-araA), which disappeared from serum in two phases, with half-lives of 31 and 114 mins. 9-beta-D-Arabinofuranosyl-2-fluorohypoxanthine (2-F-araHx) was also present in serum. After administration of 500 mg/m2 of 2-F-araAMP to mice, the apparent half-lives for the parent compound were 2.5 and 27 mins. Serum levels of 2-F-araA fell, with apparent half-lives of 36 and 185 mins. Tissue distribution studies revealed that, in mice administered 40 mg/m2, the liver, kidney, and spleen contained the highest levels of 2-F-araAMP. For most tissues, elimination of 2-F-araAMP occurred exponentially but at a slower rate than in serum. As in serum, the major tissue metabolite was 2-F-araA; other metabolites identified were 2-F-araHx, 2-fluoroadenine (2-F-A), and polyphosphorylated derivatives of 2-F-araA. In mice dosed with 500 mg/m2, the tissue distribution of metabolites was similar, but the levels were about tenfold higher. With both doses, 2-F-araAMP, 2-F-araA, and 2-F-araHx were recovered in urine. In dogs administered 40 mg/m2, serum levels of 2-F-araAMP fell rapidly in an initial phase of 5 mins and in a second phase of 30 mins. As in mice, the major serum metabolite was 2-F-araA, which disappeared in two phases of 16 and 97 mins. After administration of 500 mg/m2 to dogs, the apparent half-lives for 2-F-araAMP in serum were 9 and 51 mins. For 2-F-araA, a single phase of disappearance of 89 mins was evident. In dog serum, as compared with mouse serum, a greater percentage of the administered compound was metabolized to 2-F-araHx. During 24 hrs following administration of either 40 or 500 mg/m2 of 2-F-araAMP to dogs, less than 2% of the dose was excreted in urine as unchanged drug. Approximately equal percentages of 2-F-araA and 2-F-araHx were recovered in urine after both doses. The levels of 2-F-A in urine comprised about 2% of the radioactivity after administration of either the low or the high dose. The studies demonstrate that 2-F-araAMP undergoes rapid dephosphorylation in both mice and dogs. Deamination of the dephosphorylated product is more rapid in dogs than in mice.

Disposition of thymidine administered as large doses to rats and mice.

Blood and urine levels of thymidine and its catabolic product, thymine, have been determined for mice and rats given a single large dose of thymidine and for rats during and after infusion of large amounts of this drug. For mice given a bolus dose (400 mg/kg, 1.2 g/m2), two phases of elimination of thymidine from blood were evident, an initial phase (half-life = 4 mins) and a longer second phase (half-life = 17 mins). The initial (2-min) level in blood was 2.8 mM. For rats given an equivalent dose (150 mg/kg, 1.2 g/m2), three phases with half-lives of 2, 29, and 365 mins were observed. The initial (5-min) concentration in the blood was 0.6 mM. More of a dose of [2-14C]thymidine was converted to CO2 by rats than by mice. For both species, small amounts of radioactivity from this labeled compound became associated with macromolecules of the small intestine. Following infusion of rats with thymidine at rates of 300 and 600 mg/kg/hr for 24 hrs (60 and 120 g/m2, respectively), steady-state blood levels were approximately 0.7 and 1.5 mM, respectively. When the infusions were stopped, a phase with a half-life of 35 mins and a longer phase of indeterminate length were noted for each dose. Elimination of metabolically formed thymine from the blood of rats and excretion into the urine was mediated by a process that was apparently saturated.